Drosophila Rtf1 Functions in Histone Methylation, Gene Expression, and Notch Signaling

Drosophila Rtf1 functions in histone methylation, gene expression, and Notch signaling

Kristen Tenney*, Mark Gerber*, Anne Ilvarsonn*, Jessica Schneider*, Maria Gause*, Dale Dorsett*†, Joel C. Eissenberg*†, and Ali Shilatifard*†‡

*Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104; and †Saint Louis University Cancer Center, Saint Louis University School of Medicine, St. Louis, MO 63104

Edited by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved June 19, 2006 (received for review May 3, 2006)

The Rtf1 subunit of the Paf1 complex is required for proper monoubiquitination of histone H2B and methylation of histone H3 on lysines 4 (H3K4) and 79 in yeast Saccharomyces cerevisiae. Using RNAi, we examined the role of Rtf1 in histone methylation and gene expression in Drosophila melanogaster. We show that Dro- sophila Rtf1 (dRtf1) is required for proper gene expression and development. Furthermore, we show that RNAi-mediated reduction of dRtf1 results in a reduction in histone H3K4 trimethylation levels on bulk histones and chromosomes in vivo, indicating that the histone modiﬁcation pathway via Rtf1 is conserved among yeast, Drosophila, and human. Recently, it was demonstrated that histone H3K4 methylation mediated via the E3 ligase Bre1 is critical for transcription of Notch target genes in Drosophila. Here we demonstrate that the dRtf1 component of the Paf1 complex functions in Notch signaling.

chromatin ͉ elongation ͉ RNA polymerase II ͉ transcription ͉ monoubiquitination Fig. 1. Rtf1 regulation of histone H3 methylation. Histone H3 methylation nterplay between transcriptional activators and repressors patterns in the indicated yeast strains were tested by using Western blot Iregulates gene expression by RNA polymerase II (RNA Pol analyses with specific antibodies generated toward each modified histone H3. II). In several cases, chromatin structure is implicated in transcriptional activation and repression. Posttranslational methylation of lysines on the N-terminal tails of histones is thought to ylation and is critical for transcription of Notch target genes. In modulate higher-order chromatin folding and can activate or this study, we show that the dRtf1 component of the Paf1 repress transcription, depending on the residue being methyl- complex participates in Notch signaling in the wing margins. Our ated, the regulatory protein recruited by the methyl mark, and studies indicate that transcriptional regulation via the Paf1 whether the lysine is mono-, di-, or trimethylated (1). complex is highly conserved among eukaryotes. Histone modifications can be interdependent, such that one Results modification requires another preexisting modification (1). Hi- stone H3 methylation at lysines 4 and 79 is catalyzed by the Paf1 Complex Regulation of Histone Methylation. Many of the complex of proteins associated with Set1 (COMPASS) and subunits of COMPASS (the yeast homologue of the mammalian Dot1p, respectively in Saccharomyces cerevisiae (1–8). Methyl- MLL complex and the Drosophila trithorax complex) are re- ͞ ation at these residues requires monoubiquitination of histone quired for the proper mono-, di-, and or trimethylation of H3K4 H2B at lysine 123 by Rad6͞Bre1 (9–11). The Paf1 complex (1). In addition to COMPASS, the E2 conjugating enzyme Rad6 indirectly regulates histone methylation through its regulation of and its E3 ligase Bre1 are required for proper H3K4 methylation H2B monoubiquitination and interaction of COMPASS with via the regulation of H2B monoubiquitination (9–11). Also, it RNA Pol II (12–14). has been shown that deletion of components of the Paf1 complex ͞ The Paf1 complex in yeast is composed of five subunits, Paf1, and the Bur1 Bur2 kinase can greatly reduce histone H2B Rtf1, Cdc73, Ctr9, and Leo1, and is associated with the elon- monoubiquitination and, thereby, H3K4 methylation (12–14, 20, gating form of RNA Pol II (1, 15–18). The Rtf1 component of 21). However, deletion of RTF1, which is required for the Paf1 is required for H2B ubiquitination by Rad6 (12–14) and for activation of Rad6, seems to be required for mono-, di-, and the recruitment of Set1͞COMPASS to elongating RNA Pol II trimethylation mediated by COMPASS (Fig. 1). This observa- (12, 13). Because Rtf1 is essential for histone monoubiquitination mirrors that of effects observed when either RAD6 or tion, methylation, and transcriptional control in yeast, we sought BRE1 is deleted. Although loss of H2B monoubiquitination is the Drosophila homologue dRtf1 to characterize its role in higher not fully required for H3K4 monomethylation by COMPASS, eukaryotes. Here we use RNAi to reduce dRtf1 expression levels this observation can be explained by the fact that Rtf1 is not only and examine the in vivo effect of dRtf1 reduction on transcription required for the regulation of H2B monoubiquitination but also and development in the fly. We show that RNAi knockdown of dRtf1 causes pupal lethality. To demonstrate a role for dRtf1 in gene expression, we tested the effect of dRtf1 RNAi knockdown Conflict of interest statement: No conflicts declared. on heat shock gene expression and found that Rtf1 knockdown This paper was submitted directly (Track II) to the PNAS office. results in a reduction in heat shock (Hsp70) gene expression. Abbreviation: RNA Pol II, RNA polymerase II. Recently, Bray et al. (19) demonstrated that the Drosophila ‡To whom correspondence should be sent at the * address. E-mail: [email protected]. homologue of Bre1 is required for proper histone H3K4 meth- © 2006 by The National Academy of Sciences of the USA

11970–11974 ͉ PNAS ͉ August 8, 2006 ͉ vol. 103 ͉ no. 32 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603620103 Downloaded by guest on September 27, 2021 Fig. 3. Loss of Rtf1 levels on chromatin. SympUAST–dRtf1 ﬂies were crossed to the Actin5C–Gal4͞CyO, yϩ driver line to activate RNAi. Fixed polytene chromosome squashes were prepared from CyO (WT) (A) and Actin 5C (dRtf1 RNAi) (B) larvae and were immunostained with an antibody speciﬁc for dRtf1.

Rtf1, dRtf1 (Fig. 2A). Fig. 2B shows dRtf1 mRNA levels visualized by Northern blot during eight developmental stages. A transcript of Ϸ3 kb was detected by using dRtf1-radiolabeled antisense RNA probe. Much higher levels of transcript were observed in embryos than in any other developmental stage (Fig. 2B). In this respect, dRtf1 expression was similar to elongation factor dELL (22), but it contrasted with elongation factor dEloA expression, which is most abundant in late larvae and during pupariation (23).

dRtf1 Is Essential for Development in Drosophila. We used RNAi to lower Rtf1 mRNA and protein levels, to determine whether dRtf1 is required for Drosophila viability. RNAi knockdown of dRTF1 was accomplished by using P element-mediated trans- Fig. 2. Drosophila Rtf1 expression patterns. (A) The gene CG10955 was formation of the SympUAST vector with a 600-bp insertion of identiﬁed as the Drosophila homologue of Rtf1. (B) Total RNA was isolated dRtf1 cDNA sequence. The SympUAST vector contains two from each developmental stage of Drosophila. Fifteen micrograms of total convergent Gal4-inducible UAS promoters that can be used to RNA from each stage was loaded and analyzed by Northern blot analysis using transcribe double-stranded RNA from the dRtf1 fragment, ac- a dRtf1-speciﬁc probe. tivating the Drosophila RNAi machinery (24). SympUAST–dRtf1 flies were crossed to the Actin5C–Gal4͞CyO, yϩ driver line to activate RNAi. Rtf1 RNAi is expressed only in Actin5C–Gal4͞ plays a role in the recruitment of COMPASS to the transcribing SympUAST–dRtf1 progeny, and not their CyO, yϩ͞SympUAST– RNA Pol II (13). In addition to regulation of H3K4 methylation, dRtf1 siblings. When we used one line, 10A, 235 CyO, yϩ͞ different components of the Paf1 complex have varying effects SympUAST–dRtf1 adults were recovered with no Actin5C–Gal4͞ BIOLOGY on other types of H3 tail methylation (Fig. 1).

SympUAST–dRtf1 progeny observed. When we used a second DEVELOPMENTAL Because Paf1 is required for histone H3K4 and K79 methyl- independent line, 17C, 310 CyO, yϩ͞SympUAST–dRtf1 adults ation, we tested the effect of Paf1 loss on histone methylation were recovered with no Actin5C–Gal4͞SympUAST–dRtf1 prog- stability in vivo (data not shown). We used a tetracycline- eny observed. A large number of dead pupae were observed in regulated PAF1 gene strain grown under normal conditions, both crosses, suggesting that the Rtf1 RNAi flies were dying turning off the expression of Paf1 in the presence of tetracycline. during the pupal stage. Because lethality was observed by using Cell extracts were prepared at different time points, and histone two independent SympUAST–dRtf1 insertion lines, lethality was H3 modification stability was tested in the absence of Paf1. After unlikely to be due to ectopic gene activation at the SympUAST– approximately 4 hours in the presence of tetracycline, Paf1 levels dRtf1 insertion site. were reduced by Ͼ95%. However, even 12 hours after Paf1 loss, In Actin5C–Gal4͞SympUAST–dRtf1 third instar larvae, Rtf1 histone H3K4 and K79 methylation levels appear to be unaf- protein levels on polytene chromosomes (Fig. 3B) were reduced fected. Our observation substantiates a report by Ng et al. (14), compared with those in their CyO, yϩ͞SympUAST–dRtf1 siblings who found that H3K4 methylation seems to be a stable mark (Fig. 3A). Although Northern blot analysis showed that dRtf1 once established on the Gal gene. We verify that histone H3K4 transcripts decreased by Ϸ20% (data not shown), this level of methylation seems to be stable, further supporting a role for the reduction seems to be sufficient to cause 100% lethality. Our stability of this type of histone modification. data indicate that, upon expression of Rtf1 RNAi in Actin5C– Gal4͞SympUAST–dRtf1 progeny, the overall level of chromo- Developmental Expression of the Drosophila Rtf1, dRtf1. Loss of the somal Rtf1 was substantially reduced, suggesting that the small Rtf1 subunit of the yeast Paf1 complex has the greatest effect RNAs generated by Dicer from the long double-stranded Rtf1 on histone H2B monoubiquitination and H3K4 methylation RNA precursors may act as microRNAs (miRNAs) and may (Fig. 1). To investigate the conservation of Rtf1’s role in inhibit Rtf1 translation as well. histone methylation and regulation of gene expression in a higher eukaryote, we characterized the role of subunit Rtf1 in dRtf1 Is Required for Histone H3K4 Trimethylation on Polytene Chro- histone methylation and transcriptional regulation in Drosoph- mosomes. Because Rtf1 loss in yeast results in the abrogation of ila. CG10955 is the Drosophila structural homologue of yeast H3K4 methylation, we tested whether dRtf1 in Drosophila

Tenney et al. PNAS ͉ August 8, 2006 ͉ vol. 103 ͉ no. 32 ͉ 11971 Downloaded by guest on September 27, 2021 dRtf1 third instar larvae dRtf1 levels at the 87A and 87C loci (Hsp70 puff sites) was reduced (Fig. 5 A and B) and Hsp70 transcripts were reduced at various time points throughout a 60-min heat shock (Fig. 5C). This observation indicates that dRtf1 is required for proper regulation of heat shock gene expression in whole flies and suggests that histone methylation is required for full heat shock gene expression. A previous report showed that dRtf1 is recruited to heat shock loci in polytene chromosomes upon heat shock (25). To test whether dRtf1 recruitment is associated with H3K4 methylation at heat shock loci, we immunostained polytene chromosomes from heat shocked larvae with an antibody to trimethylated H3K4. Trimethylated H3K4 accumulates at heat shock puff sites (87A, 87C, and 93D) in response to heat shock (Fig. 5D). Note that extensive H3K4 methylation remains throughout the rest of the genome (Fig. 5D), consistent with the observation that a significant fraction of dRtf1 remains at non-heat shock loci during heat shock (25).

Knockdown of dRtf1 by RNAi Enhances Nnd-1. The Notch signaling pathway is required for regulation of cell fate decisions throughout metazoan development. Although Notch signaling is known to activate transcription of numerous target genes, little is known regarding the molecular details of this process. Recently, Bray et al. (19) reported that the Drosophila homologue of yeast Bre1 is required for Notch target gene expression in Drosophila. Because Rtf1 is also required for regulation of Rad6͞Bre1 in yeast, we tested whether dRtf1 is required for proper Notch signaling. Fig. 4. dRtf1 is required for proper histone H3K4 trimethylation. (A) Extracts Notch Nnd-1 ͞ ϩ͞ The hypomorphic allele causes limited nicking in from Actin5C–Gal4 SympUAST–dRtf1 progeny (Rtf1 RNAi On) and CyO, y the margin of the adult wing. Mutations in factors required for SympUAST–dRtf1 progeny (Rtf1 RNAi Off) were applied to SDS͞PAGE and Western blot analysis by using antibodies speciﬁc to methylated H3K4. (B and Notch signaling enhance this wing nicking; strong enhancement C) Trimethylated H3K4 levels are reduced on dRtf1 knockdown chromosomes. results in a reduced wing width. Mild, nonlethal activation of two (B) CyO, yϩ͞SympUAST–dRtf1 progeny chromosomes (Rtf1 RNAi Off) immu- independent dRtf1 RNAi knockdown transgenes, dRtf1–10A nostained with anti-trimethyl H3K4 serum (Upper) and corresponding phase- and dRtf1–17C, by an Hsp70–Gal4 driver at 27°C decreased the contrast image of the same chromosome (Lower). Trimethylated H3K4 is width of Nnd-1 wings relative to a no-RNAi control (yw) and widely distributed and enriched at sites of active transcription in WT chromo- relative to RNAi directed against dEloA (dEloA–18D) (Fig. 6). somes. (C) Actin5C–Gal4͞SympUAST–dRtf1 progeny (Rtf1 RNAi On) chromo- Both dRtf1 RNAi insertions gave significant reductions in wing somes immunostained with anti-trimethyl H3K4 serum (Upper) and corre- width relative to the dEloA and ywcontrols (P Ͻ 0.003 in all sponding phase-contrast image of the same chromosome (Lower). H3K4 cases by the Bonferroni͞Dunn test). There was no significant trimethylation is dramatically reduced in dRtf1 knockdown chromosomes. difference in wing width between ywand dEloA RNAi, dem- onstrating that the effect on Nnd-1 was not due to ectopic Gal4 functions in the same pathway. Extracts prepared from both expression. Actin5C–Gal4͞SympUAST–dRtf1 progeny (Rtf1 RNAi on) and Discussion CyO, yϩ͞SympUAST–dRtf1 progeny (Rtf1 RNAi off) were tested for methylated H3K4 levels. As shown in Fig. 4A, dRtf1 RNAi The yeast Paf1 complex interacts with RNA Pol II and regulates knockdown resulted in a significant reduction in total cellular the pattern of histone modifications; deletion of the Rtf1 subunit trimethylated H3K4. To test whether this reduction occurs in yeast has the strongest phenotype on histone H3 modification throughout the genome, fixed polytene chromosomes squashes loss (Fig. 1). Analysis of the functional homologue of the Paf1 complex from human cells indicated that this complex includes were prepared from Actin5C–Gal4͞SympUAST–dRtf1 and CyO, ϩ hCtr9, hPaf1, hLeo1, and hCdc73, and a higher eukaryotic- y ͞SympUAST–dRtf1 larvae and were immunostained with an specific subunit, hSki8 (26). The Rtf1 subunit does not appear to antibody specific for trimethylated H3K4. In WT polytene have a stable interaction with Paf1 complex from either human chromosomes, trimethylated H3K4 is widely distributed or Drosophila once purified. However, Rtf1 colocalizes broadly throughout the euchromatic chromosome arms and is highly with actively transcribing, phosphorylated RNA Pol II in a enriched at developmental puffs, sites of active transcription pattern very similar to that of Paf1 and CDC73 (25). These data (Fig. 4B). In contrast, the polytene chromosomes from dRtf1 suggest that Rft1 functions with the Paf1 complex in vivo. Our knockdown larvae consistently show reduced staining with the findings regarding the role of Rtf1 in gene expression, Notch same antibody (Fig. 4C). Together, our data suggest that the loss signaling, and fly development extend the essential role of this of Rtf1 results in the loss of H3K4 methylation in Drosophila. complex in regulating gene expression during development. Much of our understanding of gene regulation and its role in dRtf1 Affects Transcription of Heat Shock Genes. Rtf1 in S. cerevisiae development and differentiation comes from studies initially is required for COMPASS recruitment to elongating RNA Pol performed in yeast and Drosophila. The MLL gene, a frequent II (12–14), implicating it in RNA Pol II transcription. Accord- partner in chromosome translocations leading to leukemia, ingly, we tested the effect of dRtf1 reduction on transcription in exists in a large macromolecular complex, similar to its yeast flies. Because RNAi knockdown of dRtf1 in cultured S2 cells homologue SET1 in the COMPASS complex (1, 2, 27, 28). The reduces Hsp70 expression by Ϸ25% (25), we used the Hsp70 heat methylation of histones by yeast COMPASS requires ubiquiti- shock genes as reporters for the effect of dRtf1 knockdown in nation of histone H2B by the Rad6 and Bre1 proteins, and the whole animals. We found that, in Actin5C–Gal4͞SympUAST– Paf1 complex is required to activate Rad6 and Bre1 histone

11972 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603620103 Tenney et al. Downloaded by guest on September 27, 2021 Fig. 5. dRtf1 is required for proper heat shock gene expression. (A and B) CyO, yϩ͞SympUAST–dRtf1 chromosomes (Rtf1 RNAi off) (A) and Actin5C–Gal4͞ SympUAST–dRtf1 chromosomes (Rtf1 RNAi on) (B) were immunostained with anti-Rtf1 after heat shock to determine the levels of Rtf1 at heat shock puff sites upon Rtf1 RNAi induction. (C) Larvae with RNAi-mediated depletion of dRtf1 express less Hsp70 transcript than larvae that do not carry the RNAi driver construct. Male dRtf1 RNAi-carrying ﬂies were crossed to the female (Actin5C–Gal4͞CyO driver) ﬂies. The progeny were heat shocked (HS) for various times as indicated. Total RNA was prepared from both nonheat shocked and heat shocked Actin 5C–Gal4 and CyO larvae. A Northern blot probed for Hsp70 mRNA shows that transcript levels are reduced in RNAiϩ animals. rp49 mRNA was probed as a load control. (D) WT (Oregon R) chromosomes were immunostained with anti-RNA Pol II (Upper) and anti-trimethyl H3K4 serum (Lower) before and after heat shock (HS). Sites of heat shock puffs are indicated by arrows.

ubiquitination activity (9, 12, 13). Here we report that the of Notch target genes and histone H3K4 methylation (19). Work Drosophila Rtf1 subunit of the Paf1 complex facilitates Notch here indicates that Rtf1 contributes to Notch signaling and signaling, linking histone ubiquitination and methylation to gene histone methylation. Therefore, the functional connection activation by the Notch pathway. Previously, it was reported that among the Paf1 complex, Rad6͞Bre1, complex, and the histone the Drosophila homologue of Bre1 is also required for expression H3K4 methylation seen in yeast is conserved in Drosophila.In addition to revealing some of the molecular mechanisms under- lying MLL action, these observations predict that the human Paf1 complex will also play a role in Notch signaling and suggest

that the Notch pathway may be linked to the pathogenesis of BIOLOGY

leukemia via the MLL translocations. DEVELOPMENTAL Materials and Methods Plasmid Construction. The published S. cerevisiae Rtf1 sequence (GenBank accession no. NP࿝011270) was used to search the National Center for Biotechnology Information (NCBI) data- base for the Drosophila melanogaster homologue, dRtf1 (Gen- Bank accession no. NM࿝137821, gene CG10955). To create the SympUAST–dRtf1 construct, oligonucleotides were designed to PCR-amplify 600 bp (nucleotides 841–1,440) from the dRtf1 cDNA with nested EcoRI and BglII restriction sites (5Ј- TACTACAGATCTGATGCGGCTTCCACTTCAGCGGC- AGTGG-3Ј and 5Ј-TACTACGAATTCAATTTCGGCCACTC- GATACACGGGTTTC-3Ј). The amplification product was Ϸ Fig. 6. Partial knockdown of Rtf1 by RNAi increases Nnd-1 hypomorphic ligated to the EcoRI–BglII fragment ( 10.5 kb) of Sym- mutant wing phenotype severity. Two independent RNAi P element insertions pUAST–w (24). The resulting SympUAST–dRtf1 vector was used against Rtf1 (Rtf1–10A and Rtf1–17C) decreased the width of Nnd-1 wings for P element-mediated transformation as described below. An relative to no RNAi (yw) or RNAi directed against EloA (EloA–18D). The wing NCBI BLAST search of the dRtf1 600-bp fragment for short, width distributions are shown as box plots, where the horizontal lines indicate nearly exact matches indicated that there were no unintentional the 10th, 25th, 50th, 75th, and 90th percentiles. Wing width was measured RNAi targets. from the anterior margin to the posterior margin between the cross-veins, as diagrammed below the box plots. Decreased wing width indicates reduced Northern Blot Analysis. For the developmental Northern blot data, Notch function. Both Rtf1 RNAi insertions gave signiﬁcant reductions in wing ␮ width relative to the EloA and ywcontrols (P Ͻ 0.003 in all cases by the 15 g of total RNA was isolated from each developmental stage. Bonferroni͞Dunn test). There was no signiﬁcant difference in wing width To compare RNA in dRtf1 RNAi and control flies, 15 ␮g of total between ywand EloA RNAi. RNA was isolated from SympUAST–dRtf1͞Actin5C–Gal4 or

Tenney et al. PNAS ͉ August 8, 2006 ͉ vol. 103 ͉ no. 32 ͉ 11973 Downloaded by guest on September 27, 2021 SympUAST–dRtf1͞CyO, yϩ third instar larvae, respectively. For GAL4–Hsp70.PB}2 virgin females at 27°C. P{w[ϩmC] ϭ GAL4– heat shock Northern blot data, SympUAST–dRtf1͞Actin5C–Gal4 Hsp70.PB}2 provides heat shock-inducible Gal4 and was ob- ϩ or SympUAST–dRtf1͞CyO, y third instar larvae were heat tained from the Bloomington Drosophila Stock Center at Indi- shocked at 37°C for 0, 15, 30, and 45 min. ana University [stock 2077, donated by N. Perrimon (Harvard Medical School, Boston, MA)]. At 27°C with this driver, the Immunostaining of Polytene Chromosomes. Chromosomes were RNAi for Rtf1 is nonlethal. Ten randomly chosen wings from dissected from third instar larvae of the indicated genotypes and male progeny were mounted on microscope slides in Permount fixed first in Buffer A (15 mM Tris⅐HCl, pH 7.4͞60 mM KCl͞15 mM NaCl͞0.5 mM spermidine͞0.1% Triton X-100) plus 2% (Sigma) and digitally photographed and measured by using formaldehyde for 30 sec and then in 45% acetic acid plus 2% Northern Eclipse software (Empix Imaging, Mississauga, ON, formaldehyde before squashing. RNA Pol II-specific antisera Canada). Statistical analysis was performed by using Statview (Covance, Princeton, NJ) were used at 1:100 dilution, trimethyl software (SAS Institute, Cary, NC). H3K4 antisera (Upstate Biotechnology, Lake Placid, NY) were used at 1:50 dilution, dRtf1 antisera [a gift from John Lis We thank Kristy Wendt for editorial assistance. Work in D.D.’s labo- (Cornell University, Ithaca, NY)] were used at 1:200 dilution, ratory is supported by National Institute of General Medical Sciences and an FITC-coupled anti-rabbit secondary antibody was used at Grant R01 GM055683. Work in J.C.E.’s laboratory was supported by 1:100 dilution. National Science Foundation Grant MCB 0131414. Work in A.S.’s laboratory is supported by National Institutes of Health Grants Assay for Notch Signaling. ywMales or yw; P{Sym–pUAST–RNAi} 2R01CA089455 and 1R01GM069905 and by the American Cancer males with RNAi insertions were crossed to ywNnd-1; P{w[ϩmC] ϭ Society. A.S. is a Scholar of the Leukemia and Lymphoma Society.

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